An organism's DNA sequence remains fundamentally unchanged over an animal's life so that long term phenotypic and behavioral changes are thought to result from modifications in neural circuits. Recently, however, another, complementary mechanism, DNA methylation, has been implicated in these changes. DNA methylation, previously thought to act only during the imprinting of genes during development, can act quickly and reversibly on gene expression patterns in adult organisms. We propose novel experiments to test whether and where DNA methylation can lead to dramatic phenotypic and behavioral changes in vivo using a well suited cichlid fish as an animal model. In this species, A. burtoni, males exist in either of two distinct, reversible phenotypes: reproductively competent dominant (D) males and reproductively incompetent non-dominant (ND) males. The switch between phenotypes is a consequence of the social environment and produce physiological changes within minutes, ultimately resulting in remodeling of the reproductive axis. The main regulator of reproductive competence, gonadotropin releasing hormone (GnRH1) containing neurons in the pre-optic area (POA) of the brain, change their size and connectivity and there are numerous other modifications from body coloration to molecular regulation in receptor abundance. We have shown that we can induce these dramatic changes in phenotype from ND > D by global methylation and conversely from D > ND by global demethylation. In the proposed experiments, we will make genomic maps of methylation at single-base-pair resolution by combining bisulfite treatment of genomic DNA with ultra-high-throughput sequencing (Illumina Genome Analyser). The methylation state of all fish due to developmental events will be shared. However, we will identify methylation events associated with each distinct phenotype in response to natural behavioral encounters or as a result of treatment with methylation-regulating agents. These novel results will provide important insights into the relationship between methylation and behavioral phenotype. We will identify where in the brain the subset of genes implicated in social status change are located. We will also map the time course of methylation changes relative to phenotype change to discover whether these are simultaneous or sequential. The long-term objectives are to understand how methylation interacts with known brain circuitry to co-regulate behavior. The demethylating reagent we use, zebularine, is used to treat cancer and our analysis will be the first to provide a behavioral analysis of its effects. PUBLIC HEALTH RELEVANCE: Epigenetic changes in gene expression caused by methylation have been implicated in suicide, depression and other undesirable behavioral outcomes. Understanding how gene methylation alters behavior will have importance for a wide range of human behaviors.